A "protein linkage map" is a description of the multiple protein-protein interactions that exist within a cell. This map would consist of the protein complexes responsible for: processes such as transcription and replication, pathways that function in signaling and metabolism, and structures such as the cytoskeleton. Protein-protein interactions can be detected by a simple genetic assay, the two-hybrid system. This assay reconstitutes transcriptional activity by the interaction of a protein fused to a DNA-binding domain with a protein fused to a transcriptional activation domain, and has been successfully applied to a wide diversity of proteins from different organisms. In preliminary studies using the bacteriophage T7, we have demonstrated that use of the two-hybrid system can be extended to a genome-wide approach. This approach allows us to search all proteins encoded by the phage against each other to detect interacting pairs. We propose to expand this approach to generate a protein linkage map for the yeast Saccharomyces cerevisiae. This map should provide critical information concerning a multitude of yeast proteins identified by genome sequencing efforts. Furthermore, it should illuminate the organization and function of a eukaryotic cell. Specifically, we aim: (1) to generate suitable vectors, yeast reporter strains and yeast genomic libraries; (2) to screen one to two million random DNA-binding hybrids against a library of activation domain hybrids to identify protein-protein interactions; (3) to enter these interactions into a database constructed for this purpose; and (4) to use these data to derive and analyze linked sets of proteins, which should comprise various cellular pathways and structures.